PROJECT SUMMARY The proposed Phase I STTR project is a partnership between a biotechnology company KXTbio, Inc. and the University of North Carolina at Chapel Hill to develop an innovative high-throughput screening service for identifying new drug candidates that act via G protein-coupled receptors (GPCRs). This large protein superfamily has been the most successful source of therapeutic targets, accounting for 40% of all prescription pharmaceuticals in such diverse areas as cancer, cardiac dysfunction, diabetes, central nervous system disorders, obesity, inflammation, and pain. However, with much of the low hanging fruit already picked, the pace of GPCR drug discovery and development has slowed due to the challenges associated with obtaining 3D structures of GPCRs and performing structure-activity studies. Concurrently, new findings in GPCR pharmacology research have uncovered previously unknown intricacies of GPCR signaling that expanded their potential as targets for therapeutic interventions even further. Considering the decreasing productivity in the pharmaceutical industry and rising development costs, more effective methods of identifying GPCR drug candidates would be highly valuable. Although crystal structures of GPCRs are now being generated, their utility will remain limited for the foreseeable future. Various functional assays are currently the mainstay of GPCR screening efforts, and rely on detecting downstream cellular signaling events initiated by changes in GPCR activity. Chosen for their strong and easy to measure signals, these techniques are performed in live cell cultures, making them prone to off-target effects resulting in both false positives and false negatives, introducing higher variability and costs, as well as reduced throughput. KXTbio and UNC Chapel Hill aim to develop a new screening technology that would eliminate many of the drawbacks of the existing methods. The new high-throughput screening (HTS) platform will use purified GPCRs and G proteins, thus avoiding cell cultures. GPCR activity will be monitored by the immediately ensuing G protein activation involving an exchange of the bound GDP molecule for a GTP. The fluorescent analog of GTP whose fluorescent yield increases upon G protein binding will be used in the assay as the signal source. This proposed real-time HTS system can differentiate between agonists, antagonists and allosteric modulators, using a minimal number of components, thus reducing artefacts and cell-dependent effects. In contrast to related G protein activation assays it does not require filtration and washing steps or radiolabels. In this Phase I project KXTbio aims to deliver a proof of concept of the proposed HTS GPCR platform, and if successful, a Phase II project will follow, where the platform will be fully implemented, tested, and optimized in a multi-well plate format. KXTbio plans to commercialize it is as a service with a possibility of licensing the technology to drug discovery companies.